The HIV-1 Nef appears to inhibit the activation of transcription factors NF-KB and AP-1 in T-cells by interfering with the TCR-CD3 signaling pathway. The c-Raf-1 kinase integrates upstream activation signals emanating from receptor tyrosine kinases, PKC, and p21Ras with the MAP kinase and NF-KB signaling pathways, and has also been shown to activate physical association between the HIV-1 Nef protein and the c-Raf-1/Mek-1 kinase complex in vitro and in the chronically-infected CEM-SS cell line. The specific site of interaction in vitro was mapped by deletion mutagenesis to a minimal 12-amino acid sequence, Leu-His-Pro-Val-Ser-Leu-His-Gly-Met-Asp-Asp-Pro, represented by residues 165-176 at the carboxy terminal region of Nef. We found that individual or dual substitution of the conserved Asp 174 and Asp175 residues in the full-length GST-Nef sequence, completely abrogated the in vitro binding of c-Raf-1 to Nef. In a recent study, mutation of the highly-conserved di-aspartic acid motif (Asp174 and Asp175), produced the only fully stable Nef protein incapable of down-regulating the CD4 receptor. The di-aspartic acid residues are highly conserved among the different HIV-1 Nef proteins, as well as between HIV-2 and SIV Nef proteins. Furthermore, these residues (Asp/Gly-Asp-Pro-Glu-Arg-Glu 174-179), bear strong similarity to the sequence Asp-Pro-Thr-Ile-Glu-Asp (residues 33-38), at the known c-Raf-1 binding site within the effector loop of the p21Ras oncoprotein. Recent crystallographic data of the putative c-Raf-1 binding region of Nef reveals a large loop, which extends outward from the main structural domains of Nef, indicating its highly-charged residues could easily participate in protein-protein interactions. Our preliminary data also shows approximately a dozen unidentified proteins that associate directly or indirectly with Nef GST-fusion proteins. These interactions survive treatment with 300 mM NaCl, and may yield further important data regarding the interaction of Nef with cellular proteins.